PROJECT SUMMARY The goal of this research is to investigate the role of phosphorylation of the extended N-terminal domain of Mycobacterium tuberculosis SecE, an essential component of the SecYEG transmembrane protein translocation channel, in regulating protein secretion via the general secretory pathway. The SecE C-terminal domain encodes a transmembrane helix and an amphipathic helix that are highly conserved across bacteria and archaea. While SecE in many bacteria comprises only the ~65 residues that encode these features, others, such as E. coli and M. tuberculosis have an extended N-terminal domain. We have identified three Ser or Thr residues in this domain that are phosphorylated, all of which show highly significantly decreased phosphorylation in response to depletion of PknA and/or PknB, the two essential Ser/Thr kinases of this pathogen. These findings provide the premises for our hypothesis that the SecE extended N-terminal domain regulates protein secretion in a phosphorylation-dependent manner. To investigate the regulatory role of this domain, we propose two Aims. In Aim 1 we will perform quantitative proteomics of culture filtrates of PknA replete and PknA depleted M. tuberculosis strains. We will also construct strains expressing native SecE or SecE with phosphomimetic or phosphoablative substitutions at the identified phosphorylation sites, and quantify protein secretion in these strains. These experiments will determine the effects of SecE N-terminal domain phosphorylation on protein secretion. In Aim 2 we will identify proteins that interact with SecE using two complementary methods, i) co- immunoprecipitation with SecE in M. tuberculosis and ii) two-hybrid assays to detect protein-protein interactions with native, phosphomimetic and phosphoablative SecE. These experiments will suggest how SecE and its phosphorylation may function in the general secretion pathway. We anticipate that this research will reveal a novel mechanism for regulating protein secretion that will be relevant for tuberculosis pathogenesis and may lead to new therapeutic approaches to treat this disease.